Background. Neurotrophin (NT) proteins control some of the most fundamental neurological processes through interactions with their cognate Trk receptors. Activation of a Trk by an NT results in phosphorylation of the intracellular tyrosine residues and triggers downstream signaling pathways that mediate neurite outgrowth, neuronal differentiation or survival. It is known that the primary site of interaction between human NT-4/5 and its cognate Trk receptor takes place at the immunoglobulin-like domain 5 in the extracellular portion of the Trk receptor (TrkB-d5), but there is also evidence that the long flexible linker region spanning the Trk sequence between domain 5 and the transmembrane segment of the receptor may also play a critical role in molecular recognition. Proteins involved in signal transduction often require structural flexibility to function properly or interact successfully with multiple targets. Evidence that these motions could be critical components to binding selectivity has been provided by observations that portions of NT-4/5 undergo disorder-to-order transitions upon binding TrkB-d5 and that both proteins display significant conformational exchange alone and in complex. Specific aims. The proposal outlines a plan to pursue a thorough, atomic-level characterization of this NT/Trk interaction and the role of protein motions in binding selectivity and molecular recognition using Nuclear Magnetic Resonance (NMR) spectroscopy as our primary analytical tool. Our specific aims are to 1) examine the extent of the interactions between NT-4/5 and the linker region C-terminal to domain 5 on TrkB and identify specific contacts between them, 2) measure and compare the backbone dynamics, on the ps-ns and ms timescales, of isotopically labeled hNT-4/5 in its unbound state, and when bound to unlabeled hTrkB-d5 or hTrkB- d5L (TrkB domain 5 with attached linker region), and 3) measure and compare the backbone dynamics of isotopically labeled hTrkB-d5 or hTrkB-d5L in its unbound state, and when bound to unlabeled hNT-4/5. NMR experiments will include titrations to monitor chemical shift changes upon complex formation, residual dipolar coupling and hydrogen exchange experiments to structurally characterize the TrkB/NT interactions, and spin relaxation experiments to analyze protein motions in NT-4/5 and TrkB at multiple timescales. Health-related significance. Mutations and modifications to NTs have been linked to numerous illnesses, including Alzheimer's and Parkinson's disease, chronic pain and arthritis. There is widespread interest in developing NT-based therapeutics to treat these conditions, but success is dependent on having a comprehensive grasp of the mechanism of specificity and function inneurotrophins in order to yield selective drugs with fewer side effects. The results of these studies will be the first to provide insight into the correlation between protein motions and binding selectivity in NTs and may ultimately be critical for furthering our understanding of the bio- physical properties of this signaling process and improving our ability treat neurological diseases. PUBLIC HEALTH RELEVANCE: Our ultimate goal is to understand, at the most fundamental molecular level, the connection between internal motions within a protein involved in signal transmission and its ability to recognize and interact with another protein that is participating in the signaling pathway. If we can contribute to an improved and more sophisticated understanding of this process, we may be better able to treat neurological diseases such as Parkinson's, Alzheimer's or depression using drugs that have been designed to have fewer negative side effects.